Evolution of an Escherichia coli Protein with Increased Resistance to Oxidative Stress

L-1,2-Propanediol:NAD1 1-oxidoreductase of Escherichia coli is encoded by the fucO gene, a member of the regulon specifying dissimilation of L-fucose. The enzyme normally functions during fermentative growth to regenerate NAD from NADH by reducing the metabolic intermediate L-lactaldehyde to propanediol which is excreted. During aerobic growth L-lactaldehyde is converted to L-lactate and thence to the central metabolite pyruvate. The wasteful excretion of propanediol is minimized by oxidative inactivation of the oxidoreductase, an Fe21-dependent enzyme which is subject to metalcatalyzed oxidation (MCO). Mutants acquiring the ability to grow aerobically on propanediol as sole carbon and energy source can be readily selected. These mutants express the fucO gene constitutively, as a result of an IS5 insertion in the promoter region. In this study we show that continued selection for aerobic growth on propanediol resulted in mutations in the oxidoreductase conferring increased resistance to MCO. In two independent mutants, the resistance of the protein was respectively conferred by an Ile7 3 Leu and a Leu8 3 Val substitution near the NAD-binding consensus amino acid sequence. A site-directed mutant protein with both substitutions showed an MCO resistance greater than either mutant protein with a single amino acid change.This work was supported by United States Public Health Service Grants GM40993 and GM30693 from the NIGMS and Grant PB94-0829 from the Dirección General de Investigación Científica y Técnica, Madrid, Spain. Help from the Comissionat per Universitats i recerca de la Generalitat de Catalunya and the Ajuntament de Lleida was also received